Treatment for infection with hepatitis b virus alone or in combination with hepatitis delta virus and associated liver diseases

ABSTRACT

The invention concerns the use of cyclophilin inhibitors in the treatment of Hepatitis B and Hepatitis D virus infections.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates to non-immunosuppressive cyclosporinanalogues which bind to cyclophilins, which are cyclophilin inhibitors,in particular to their pharmaceutical use in the treatment of infectionwith Hepatitis B virus (HBV) alone or in combination with HepatitisDelta virus (HDV) and liver diseases caused by such infections.

The cyclosporins and the non-immunosuppressive analogues comprise aclass of structurally distinctive, cyclic, poly-N-methylatedundecapeptides, commonly possessing pharmacological, in particularimmunosuppressive, or anti-inflammatory activity. The first of thecyclosporins to be isolated was the naturally occurring fungalmetabolite Ciclosporin or Cyclosporine, also known as cyclosporin A(CsA). Cyclosporins which bind strongly to cyclophilin but are notimmunosuppressive have been identified. PCT/EP 2004/009804, WO2005/021028, or WO 2006/071619 (which are incorporated by referenceherein in their entireity) disclose non-immunosuppressive cyclosporinswhich bind to cyclophilin and have also been found to have an inhibitoryeffect on Hepatitis C virus (HCV). WO 2006/038088, incorporated hereinby reference in its entirety, describes methods and compositions for theuse of alisporivir in the treatment of HCV. Alisporivir (DEB025 orDebio-025) is a cyclophilin (Cyp) inhibitor and its mode of action as ananti-HCV agent is via inhibition of host proteins, in particular ofcyclophilin A, that are directly involved in HCV replication.

Hepatitis B virus (HBV) is the smallest human DNA virus (1). The HBVgenome is partially double-stranded, circular DNA with overlappingreading frames that encode the HBV proteins: envelope proteins—i) small,Hepatitis B surface antigen (HBsAg); ii) middle—preS2 plus HBsAg; iii)large—preS1 plus preS2 plus HBsAg; nucleocapsid protein, hepatitis Bcore antigen (HBcAg). Hepatitis B e antigen (HBeAg) is a non-structuralprotein produced during the HBV replication which shares 90% amino acidswith the nucleocapsid HBcAg. Eight genotypes of HBV, designated A to H,have been determined, each having a distinct geographical distribution.The virus is non-cytopathic, with virus-specific cellular immunity beingthe main determinant for the outcome of exposure to HBV—acute infectionwith resolution of liver diseases with 6 months, or chronic HBVinfection that is frequently associated with progressive liver injury.Detection of HBsAg in the serum by conventional diagnostic immunoassays,is the key diagnostic marker for infection with HBV and persistentdetection of HBsAg in serum for more than 6 months is the hallmarkchronic HBV infection (2,3,4). The best marker for clinicallysignificant HBV replication is the level of HBV DNA in serum, asdetected by sensitive polymerase chain reaction (PCR)-based assay.

Worldwide more than 350 million people are chronically infected with HBVand are thus at increased risk of developing serious liver disease—suchas chronic hepatitis, cirrhosis, liver failure and hepatocellularcarcinoma (HCC). The natural evolution of chronic HBV infection includesfour consecutive phases: (1) early ‘immunotolerant’ phase—high levels ofvirus replication and minimal liver inflammation; (2) immune reactivephase—significant hepatic inflammation and elevated serumaminotransferases; with some patients progressing to (3)‘non-replicative’ phase—seroconversion to anti-HBe; undetectable or lowlevel of viremia (below 2000 IU/ml by PCR-based assays); resolution ofhepatic inflammation; and (4) HBeAg-negative chronic hepatitis B—due tothe emergence of specific viral mutations, which prevent the productionof HBeAg but do not hamper virus replication. This form of CHB ischaracterized by fluctuating serum HBV DNA and serum aminostransferases(ALT and AST) levels, and progressive liver disease. It is important tonote that chronic hepatitis B (CHB) may present either as hepatits B eantigen (HBeAg)-positive or HBeAg-negative CHB. Longitudinal studies ofpatients with CHB indicate that the 5-year cumulative incidence ofdeveloping cirrhosis ranges from 8 to 20%. The 5-year cumulativeincidence of hepatic decompensation is approximately 20% (2). Theworldwide incidence of HCC has increased and presently constitutes thefifth most common cancer (2,3,4). The annual incidence of HBV-relatedHCC is high ranging from 2-5% when cirrhosis is established (2).

The primary goal of treatment for CHB is to permanently suppress HBVreplication and improve liver disease. Clinically important short-termgoals are to achieve HBeAg-seroconversion, normalization of serum ALTand AST, resolution of liver inflammation and to prevent hepaticdecompensation (2). The ultimate goal of treatment is to achieve durableresponse to prevent development of cirrhosis, liver cancer and prolongsurvival. HBV infection can not be eradicated completely due topersistence of a particular form of viral covalently closed circular DNA(ccc HBV DNA) in the nuclei of infected hepatocytes. However,treatment-induced clearance of serum HBsAg is a marker of termination ofchronic HBV infection and has been associated with the best long-termoutcome (2,3).

Seven drugs are currently available for treatment of CHB—conventionalinterferon, pegylated interferon and direct antiviral agents. The directantivirals (nucleos/tide analogues) belong to three classes:L-nucleosides (lamivudine, telbivudine and emtricitabine);deoxyguanosine analogue (entecavir) and nucleoside phosphonates(adefovir and tenofovir) which directly interfere with HBV DNAreplication, primarily as chain terminators (2,3,4). In HBeAg-positivepatients, virological treatment response rates (undetectable serum HBVDNA) at one year are when treated with: peg-interferon 25%; lamivudine36-40%; entecavir 67%; telbivudine 60%; tenofovir 74% (2). Loss of HBsAgafter one year is very low—between 0 and 3%. In HBeAg-negative patientswith CHB the rates of undetectable HBV DNA at one year are when treatedwith: peg-interferon 63%; lamivudine 72%; entecavir 90%; telbivudine88%; tenofovir 91%. Loss of HBsAg was 0% with any of the directantiviral agents. The currently available treatments are suboptimal andthere is a need for better therapies to meet the treatment goals in CHB.The key limitations for interferon treatment are major side-effects, lowrate of HBV DNA suppression and low rate of ALT normalization; keylimitations of the treatment with direct antivirals are: development ofresistance; rebound of HBV replication after stopping therapy requiringprolonged, life-long therapy, very low rate of HBsAg clearance,increasing the risk of adverse events with prolonged, life-long therapy.

A proportion of patients with chronic HBV infection (serumHBsAg-positive) will have co-infection with Hepatitis Delta virus (HDV).HDV is a defective virus with a single-stranded circular RNA genome,causing acute or chronic liver diseases only in association withhepatitis B virus (1). The only protein expressed by the HDV RNA ishepatitis Delta antigen, which is the nucleocapsid of the virus. HDVdoes not encode its own envelope protein (HBsAg) and must “borrow” thisfrom hepatitis B virus. Thus, the infection with HDV occurs only in thepresence of HBV—either simultaneously with HBV (co-infection) orsubsequently, with the duration of infection determined by that of HBsAgpositivity. In a HBsAg-positive patient, active infection with HDV isconfirmed by the detection of HDV RNA in serum, immunohistochemicalstaining for Hepatitis Delta antigen in hepatocytes, or detection of IgManti-HDV in serum (2).

HDV has worldwide distribution with the highest endemicity inMediterranean countries, parts of south and middle Africa and in SouthAmerica. Is it estimated that 5% of all hepatitis B virus carriers areinfected with HDV (5). In countries where HBV prevalence is low, such asNorth America and northern Europe, HDV infection is largely restrictedto populations at risk for parenteral HBV exposure like intravenous drugusers Immigration from regions of high HDV endemicity has increased theprevalence in several European countries recently leading approximately10% amongst chronic HBV patients.

The only approved treatment of chronic hepatitis D is interferon-alphawith unsatisfactory results. Addition of ribavirin to interferon did notimprove the response. Direct antivirals that block HBV replication wereshown to have no effect on HDV replication. Combinations of interferonplus lamivuidne or interferon plus adefovir did not improve the responsecompared to interferon alone (2,3,6). Thus, treatment of chronichepatitis D remains a major unmet medical need as HDV-induced liverdamage leads to cirrhosis, liver decompensation and in some cases deathdue to liver failure.

Therefore it is an object of the present disclosure to provide newmethods for the treatment of patients with hepatitis B virus infectionalone or patients infected with hepatitis B virus and hepatitis Deltavirus and the liver diseases or disorders induced by these infections,such as chronic hepatitis, cirrhosis, hepatic decompensation and HCC.

Surprisingly we have found that cyclophilin inhibitors, in particularalisporivir, have antiviral properties against Hepatitis B virus thatcan be used effectively in the treatment of HBV infections. Inparticular, we have found that alisporivir abrogates (or interferes) HBVreplication and reduces HBsAg production in liver cells.—Furthermore,the cyclophilin inhibitors, in particular alisporivir, can also be usedin the treatment of chronic hepatitis D.

Accordingly, the present invention provides new anti-HBV and anti-HDVtreatments using alisporivir.

Furthermore, the present disclosure provides methods for the treatmentof HBV and/or HDV infections and of the induced liver diseases inpatients comprising administering an effective amount of a cyclophilininhibitor, in particular alisporivir, either alone or in combinationwith a direct antiviral agent or with interferon-alfa. The disclosurefurther provides alisporivir for use in the treatment of HBV infectionand associated liver diseases and also for use in the treatment ofpatients infected with HBV and HDV and associated liver disease.

SUMMARY OF THE DISCLOSURE

Further the following is described:

1.1 A method for treating Hepatitis B virus infections and HBV-induceddisorders in a patient, comprising administering to said patientalisporivir.

1.2 A method for inhibiting HBV replication and HBsAg production,comprising administering alisporivir.

1.3. A method for treating Hepatitis Delta virus infections andHDV-induced disorders in a patient, comprising administering to saidpatient alisporivir.

1.4. A method for inhibiting HDV replication and HBsAg production,comprising administering alisporivir.

1.5. A method for reducing HBV or HDV-induced liver damage, and forreduced liver cell death comprising administering alisporivir.

1.6. A method for normalization of serum ALT levels comprisingadministering alisporivir.

1.7. A method for reduction or resolution of liver inflammationcomprising administering alisporivir.

2. Use of alisporivir in the preparation of a pharmaceutical compositionfor use in any method as defined above.

3. Use of alisporivir in the preparation of a medicament for use in anymethod as defined above.

4. Use of alisporivir in combination with a direct antiviral agent thatinhibits HBV replication in the preparation of a medicament for use inany method as defined above.

5. Use of alisporivir in combination with an interferon for treatment ofpatients with HBV infections and associated liver disease, and forpatients infected with HBV and HDV and associated liver disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the significant reduction of intracellular HBV DNAlevels (by 10-fold at 72 hours) was observed with alisporivir at aconcentration of 5 micrograms/mL.

As it can bee seen in FIG. 2, alisporivir at a concentration of 5.0micrograms/mL showed a greater effect in reducing the HBV DNA levels incell culture supernatants when compared with the effect of NIM811.

FIG. 3 illustrates the dose-dependent reduction of HBV DNA secreted fromtransiently transfected cells (Huh7) or from stably transfected(HepG2215) cells in presence of DEB025.

FIG. 4 illustrates dose dependent reductions of cytoplasmic HBV DNA inHuh7 and HepG2215 cells in presence of DEB025.

FIG. 5 illustrates reductions of secreted HBV DNA in HepG2215 cells inpresence of DEB025 (DEB), telbivudine (TELB) and DEB+TELB.

FIG. 6 illustrates reductions of cellular HBV DNA in HepG2215 cells inpresence of DEB025 (DEB), telbivudine (TELB) and DEB+TELB.

DESCRIPTION OF THE DISCLOSURE

As used herein a “Hepatitis B virus infected patient” means a patientbeing infected with any Hepatitis B virus genotype, e.g., genotype A, B,C, D etc. In some embodiments, the patient is infected with Hepatitisdelta virus. In some embodiments, the Hepatitis B virus infected patientmay be infected also with Hepatitis delta virus.

In the present invention, an interferon may be pegylated ornon-pegylated and may include interferons such as: Intron-A®, interferonalfa-2b (Schering Corporation, Kenilworth, N.J.); PEG-Intron®,peginteferon alfa-2b (Schering Corporation, Kenilworth, N.J.); Roferon®,recombinant interferon alfa-2a (Hoffmann-La Roche, Nutley, N.J.);Pegasys®, peginterferon alfa-2a (Hoffmann-La Roche, Nutley, N.J.);Berefor®, interferon alfa 2 available (Boehringer IngelheimPharmaceutical, Inc., Ridgefield, Conn.); Sumiferon®, a purified blendof natural alpha interferons (Sumitomo, Japan); Wellferon®,lymphoblastoid interferon alpha n1 (GlaxoSmithKline); Infergen®,consensus alpha interferon (InterMune Pharmaceuticals, Inc., Brisbane,Calif. and Amgen, Inc., Newbury Park, Calif.); Alferon®, a mixture ofnatural alpha interferons (Interferon Sciences, and Purdue FrederickCo., Conn.); Viraferon®; and combinations of these interferons.

Conjugated interferons that may be used include, for example, Albuferon(Human Genome Science) which is conjugated to human albumin. Interferonconjugated to a water-soluble polymer or polyalkylene oxide homopolymerssuch as polyethylene glycol (PEG) or polypropylene glycols,polyoxyethylenated polyols, copolymers thereof and block copolymersthereof. As an alternative to polyalkylene oxide-based polymers,effectively non-antigenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-basedpolymers and the like can be used. Interferon-polymer conjugates aredescribed in U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,917,888, EPA 0 236987, EPA 0 510 356 and WO 95/13090. Since the polymeric modificationsufficiently reduces antigenic responses, the foreign interferon neednot be completely autologous. Interferon used to prepare polymerconjugates may be prepared from a mammalian extract, such as human,ruminant or bovine interferon, or recombinantly produced. Other forms ofinterferons include interferon beta, gamma, tau and omega, such as Rebif(Interferon beta 1a) by Serono, Omniferon (natural interferon) byViragen, or Omega Interferon by Boehringer Ingelheim. Oral interferonssuch as oral interferon alpha by Amarillo Biosciences.

Additional examples of interferons that may be used include pegylatedinterferon alpha, for example pegylated interferon α-2a, pegylatedinterferon α-2b, pegylated consensus interferon or pegylated purifiedinterferon-α product. Pegylated interferon α-2a is described in EuropeanPatent 593,868 (incorporated herein by reference in its entirety) andcommercially available e. g. under the trade name PEGASYS® (Hoffmann-LaRoche). Pegylated interferon-α-2b is described, e.g. in European Patent975,369 (incorporated herein by reference in its entirety) andcommercially available e.g. under the trade name PEG-INTRON A® (ScheringPlough). Pegylated consensus interferon is described in WO 96/11953(incorporated herein by reference in its entirety).

In preferred embodiments, the interferon used in the methods of theinvention is pegylated interferon. In other embodiments, the interferonis selected from the group consisting of interferon alpha-2a, Interferonalpha-2b, a consensus interferon, a purified interferon alpha product ora pegylated interferon alpha-2a, pegylated interferon alpha-2b, andpegylated consensus interferon, a mixture of natural alpha andcombinations thereof.

Preferably the methods using interferon alpha use a pegylated interferonalpha-2b and the amount of pegylated interferon alpha-2b is from 0.5 to2.0 micrograms/kilogram per week on a weekly, three times a week, everyother day or daily basis.

As used herein, “microgram/kilogram” means microgram drug per kilogrambody weight of the mammal—including man—to be treated.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventative treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

By “therapeutic regimen” is meant the pattern of treatment of anillness, e.g., the pattern of dosing used during HBV therapy. Atherapeutic regimen may include an induction regimen and a maintenanceregimen.

The phrase “induction regimen” or “induction period” refers to atherapeutic regimen (or the portion of a therapeutic regimen) that isused for the initial treatment of a disease. The general goal of aninduction regimen is to provide a high level of drug to a patient duringthe initial period of a treatment regimen. An induction regimen mayemploy (in part or in whole) a “loading regimen”, which may includeadministering a greater dose of the drug than a physician would employduring a maintenance regimen, administering a drug more frequently thana physician would administer the drug during a maintenance regimen, orboth.

The phrase “maintenance regimen” or “maintenance period” refers to atherapeutic regimen (or the portion of a therapeutic regimen) that isused for the maintenance of a patient during treatment of an illness,e.g., to keep the patient in remission for long periods of time (monthsor years). A maintenance regimen may employ continuous therapy (e.g.,administering a drug at a regular intervals, e.g., weekly, monthly,yearly, etc.) or intermittent therapy (e.g., interrupted treatment,intermittent treatment, treatment at relapse, or treatment uponachievement of a particular predetermined criteria [e.g., pain, diseasemanifestation, etc.]).

As used herein, the term “about”, unless the context dictates otherwise,is used to mean a range of + or −10%.

In other embodiments, the interferon alpha is a pegylated interferonalpha-2a and the amount of pegylated interferon alpha-2a administered isfrom 20 to 250 micrograms/kilogram per week on a weekly, three times aweek, every other day or daily basis. Preferably, the interferonpeg-IFNa2a is administered at an amount of 180 micrograms once per week.

In specific embodiments, the exemplary interferon used in the methodsherein is interferon selected from the group consisting of Intron-A®;PEG-Intron®; Roferon®; Pegasys®; Berefor®; Sumiferon®; Wellferon®;Infergen®; Alferon®; Viraferon®; Albuferon® (Human Genome Science);Rebif; Omniferon; Omega and combinations thereof.

In some embodiments, alisporivir is used in the treatment of Hepatitis Bvirus infection in a patient and/or in the treatment of Hepatitis Dvirus infection in a patient. In still another aspect, alisporivir isadministered in combination with a direct antiviral agent or aninterferon.

In some other embodiments it is described a method for treating aHepatitis B virus infection in a patient comprising administering aneffective amount of alisporivir and optionally administering to thepatient an interferon or a direct antiviral agent. In still an otheraspect, alisporivir is for use in improvement of liver inflammation,reduce liver cell death (as assessed by ALT levels) and prevention ofprogression of liver disease.

In another embodiment, a pharmaceutical composition comprisingalisporivir for use according to any of the methods disclosed herein anda package comprising said pharmaceutical composition in combination withinstructions to administer said composition is described.

In another embodiment, alisporivir may be administered with additionalagents of the standard of care that promote the antiviral efficacy ofthe therapy treatment.

Direct antiviral agent, is used herein to mean agents that interferewith specific steps in the hepatitis B virus (HBV) replication cycle. Adirect antiviral agent that inhibits HBV replication may be for exampleany of the currently anti-HBV agents approved for the treatment of HBV,i.e. telbivudine, lamivudine, emtricitabine, entecavir, adefovir andtenofovir.

In treatment described above effective dosages of the standard of careagents are administered in compositions, i.e. they may be administeredtogether (i.e., simultaneously), but may also be administered separatelyor sequentially. In general, combination therapy is typicallyadministered together, the rationale being that such simultaneousadministration induces multiple simultaneous stresses on the virus. Thespecific dosages given will depend on absorption, inactivation andexcretion rate of the drugs as well as other factors. It is to be notedthat dosage values will also vary with the severity of the condition tobe alleviated. The terms “co-administration” or “combinedadministration” or “administered in combination with” or the like asutilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time. Fixedcombinations are also within the scope of the present disclosure. Theadministration of a pharmaceutical combination of the disclosure resultsin a beneficial effect, e.g. a synergistic or additive therapeuticeffect, compared to a monotherapy applying only one of itspharmaceutically active ingredients or as compared to the currentstandard of care therapy. The treatment used in the methods describedherein may be administered by any conventional route. One or morecomponents may be administered parentally, e.g., in the form ofinjectable solutions or suspensions, or in the form of injectabledeposit formulations. Preferably, alisporivir will be administeredorally in the form of capsules, tablets or solutions or suspensions fordrinking. Pharmaceutical compositions for oral administration comprisingalisporivir typically further comprise one or more pharmaceuticallyacceptable carrier substances. Typically, these compositions areconcentrated and need to be combined with an appropriate diluent, e.g.,water, prior to administration. Pharmaceutical compositions forparenteral administration typically also include one or more excipients.Optional excipients include an isotonic agent, a buffer or otherpH-controlling agent, and a preservative. These excipients may be addedfor maintenance of the composition and for the attainment of preferredranges of pH (about 6.5-7.5) and osmolarity (about 300 mosm/L).

The administration of alisporivir as described herein is in a singledose form or in more than one dosage form; one or more oral dosage formsmay be administered at each time per day. In some embodiments,alisporivir is administered in doses of 200 mg to 1000 mg.

The efficacy of the therapy regimen may be monitored using standardprotocols. Treatment may be followed by determinations of HBV levels inserum and measurement of serum ALT levels. For example, the patients maybe assessed for the presence of HBV DNA in their serum. HBV DNA (IU/mL)can be measured at regular intervals during the treatment, e.g., at Day1 (pre-dose and 4, 8, and 12 hours post-dose) and pre-dose at Day 2, Day3, Day 8, Day 15, Day 29, and at Week 12, Week 24, Week 36, Week 48,Week 72 (when applicable), and at follow up.

The efficacy of therapy will be monitored using internationally acceptedparameters (2,3,4):

1.1. Monitoring Response in Patients with Hepatitis B Virus

-   -   a) Serum HBV DNA levels are monitored using sensitive        quantitative PCR-based assays to assess the effect on viral        replication.    -   b) In HBeAg-positive patients—HBeAg is monitored along with the        corresponding anti-HBe to determine whether HBe-seroconversion        has occurred.    -   c) Serum levels of ALT and/or AST are monitored to assess impact        on liver inflammation and liver cell death    -   d) Serum HBsAg is monitored—qualitatively and quantitatively as        HBsAg clearance would indicate optimal treatment outcome.    -   e) The development of mutations in the HBV genome that confer        resistance to the treatment used        1.2 Monitoring Response in Patients with Hepatitis Delta Virus.        In Addition to the Above Listed Markers for HBV Infection, the        Following HDV-Specific Test will be Used:    -   a) Serum HDV RNA levels are monitored to assess the effect on        hepatitis Delta virus replication.    -   b) Detection of Delta antigen by immunohistochemitry in liver        tissue may provide additional information indicating ongoing        HDV-replication.

The following Examples illustrate the invention described hereinbefore.

EXAMPLES Experimental Design and Human Cell Lines:

The experiments have used several human hepatoma-derived cell lines thathave been established as models for in vitro studies on hepatitis Bvirus or hepatitis delta virus life cycle and to evaluate the impact ofvarious compounds on viral replication and viral proteinproduction—HepG2 and HuH-7 (HBV negative), PLC/PRF/5 (HBV positive,producing HBsAg only), and HepG2.2.15 (HBV positive, supporting full HBVreplication).

A) Investigation of Antiviral Activities of Cyclophilin Inhibitors, inParticular DEB025 (Alisporivir), on Hepatitis B Virus Replication andHBsAg Production.

HepG2215 cell line, derived from HepG2 cells, was stably transfectedwith HBV DNA and supported full HBV replication with production ofinfectious virions and HBsAg. HepG2215 cells were cultured in 12-wellplates, as described previously (7, 8), for 7 days prior to adding thecyclophilin inhibitors.

The cells were treated with 0.25, 1.0 or 5.0 micrograms/mL of NIM811 orDEB025; cells and supernatants were harvested separately at baseline, 6,24, 48 and 72 hours after the addition of NIM811 or DEB025,respectively.

To assess the role of cyclophilin A in HBV replication, HepG2215 cellswere transfected with siRNA for cyclophilin A and incubated with mediumonly or with the addition of NIM811 or DEB025.Similarly, the role ofCyclophilin B, C and D in HBV and HDV replication will be evaluated bysiRNA specific for CypB, C and D.

HuH-7 cells were transfected with a plasmid containing a pBlueScriptKS(+) vector with a 1.5 HBV DNA genomes of subtype adw2 was used.Subconfluent HuH-7 cells were transfected with 5 μg of plasmid DNA per60-mm-diameter dish using 10 μl of Superfect reagent (Qiagen, Crawley,UK). The supernatants were replaced 18 h posttransfection with mediumsupplemented with 10% FCS, and the cells were cultured for 3 days, asdescribed (7).

PLC/PRF/5 cells (Alexander cell line) contains integrated fragment ofHBV DNA and produces HBsAg only, but does not support full HBVreplication (9). This is analogous to the hepatocytes in patients thatare inactive HBsAg carriers (i.e the non-replicative phase of chronicHBV infection). This cell line was employed to assess specifically theimpact of cyclophilin inhibitors on HBsAg production separate from fullvirion production. Results

-   -   1) FIG. 1: Both cyclophilin inhibitors NIM811 and DEB025 reduced        intracellular, HBV nucleocapsid-associated HBV DNA levels. The        HBV DNA reduction was between 2 and 10-fold as compared to the        HepG2215 cells in the absence of NIM811 or DEB025. The most        pronounced reduction of intracellular HBV DNA levels (by 10-fold        at 72 hours) was observed with DEB025 (5 micrograms/mL), which        was greater than the reduction observed with NIM811 at 72 h.    -   2) FIG. 2: DEB025 (at 5.0 micrograms/mL) showed a greater effect        in reducing the HBV DNA levels in cell culture supernatants when        compared with the effect of NIM811.

B) Investigation of Antiviral Activities of Cyclophilin Inhibitors, inParticular DEB025 (Alisporivir), on Hepatitis Delta Virus Replication.

Infection with the Hepatitis delta virus (HDV) is always associated withthe presence of the helper virus—hepatitis B virus. The basis for thisdependence is that the HBV envelope proteins are required for both HDVentry into hepatocytes and the assembly of new HDV particles. Onceinside a susceptible cell, the single-stranded circular RNA genome ofHDV can replicate by RNA-directed RNA synthesis using redirection of ahost RNA polymerase. In vitro, it is possible to study HDV genomereplication in cultured cell lines in the absence of the helper HBV. Forexample, replication is initiated when cells are transfected with a cDNAversion of the HDV sequence (10). In addition, the need can be met bycotransfecting the HDV RNA along with an mRNA for δAg (11).

C) Investigation of Antiviral Activities of Cyclophilin Inhibitors, inParticular DEB025 (Alisporivir) in Combination a Direct Anti-ViralTargeting HBV-DNA Polymerase (Telbivudine), on Hepatitis B VirusReplication.

Stably transfected (HepG2215) and transiently transfected (HUH-7) cells,producing full

HBV virions and HBsAg particles, were treated with a range ofAlisporivir concentrations (0.25, 1.0 5.0 or 20 micrograms/ml) alone,telbivudine alone, or combinations of Alisporivir and telbivudine. Todetermine the involvement of individual cyclophilins, HepG2215 cellswere transfected with siRNA-specific for cyclophilin (Cyp) A, C or D andadditionally treated with Alisporivir. Cytoplasmic extracts andsupernatants were harvested at baseline; 24, 48 and 72 hourspost-treatment. The kinetics of antiviral activity was assessed byquantitation of intracellular and secreted HBV-DNA (real-time qPCR) andHBsAg levels (ELISA).

Both in HepG2215 and HUH-7 cells, Alisporivir treatment resulted indose-dependent reduction of intracellular and secreted HBV-DNA at alltime points, by 70% (p=0.004) and 63% (p<0.001), respectively, comparedwith untreated controls. The combination of

Alisporivir and telbivudine had greater effects in reducingintracellular (p=0.001) and secreted (p=0.028) HBV-DNA, and >3-foldreduction of HBsAg versus either Alisporivir or telbivudine alone. CypA,C or D expression was markedly reduced after transfection withcorresponding siRNA, which was associated with significant decrease ofHBV-DNA and HBsAg levels (p<0.001). Alisporivir treatment of cellssilenced for CypA, C or D further reduced HBV-DNA and HBsAg levels, withgreater antiviral effects in CypC or CypD silenced cells, compared withCypA silenced cells (p<0.001).

These results suggest that Alisporivir interferes with multiple sites ofHBV replication and its antiviral activity is synergistic with directantiviral targeting viral DNA polymerase, such as telbivudine.

1. Cell Culture and Transfection.

The human hepatoma cell line HuH7 was cultured in Dulbecco's modifiedEagle's medium supplemented with 10% fetal bovine serum, penicillin, andstreptomycin and was incubated at 37° C. in 5% CO₂, as describedpreviously (12). For transfection studies, cell cultures were seededovernight in six-well plates or 60-mm-diameter petri dishes andtransfected with 5 and 10 μg, respectively, of 50:50 mixtures of HDAgmRNA and HDV RNA (1.2 times the genome-length) using DMRIE-C reagent(Gibco BRL) according to the manufacturer's directions. Followingtransfection, the cultures were incubated overnight, the medium waschanged, and incubation was continued for up to an additional 2 to 7days.

2. HBV DNA Quantitation in Cytoplasm and Cell Supernatants

HBV replication was assessed by quantitation of both viralnucleocapsid-associated HBV DNA (cytoplasmic) and the HBV DNA levels incell culture supernatants, as described previously (8,9). NucleocapsidHBV DNA was extracted and quantitated by TaqMan real-time PCR (ABI Prism7700). Intracellular HBV DNA was normalized with b-actin as ahouse-keeping gene. Secreted HBV DNA was extracted with QIAmp DNAminikit (Qiagen, Sussex, UK) and also quantitated by TaqMan. Thesensitivity of HBV DNA quantitation was verified in each run with serialdilutions of HBV DNA plasmid, as well as the EuroHep HBV DNA standard,as described (9,10).

3. HBsAg Quantitation

HBsAg levels in cell culture supernatants (from 2215 and PLC/PRF/5cells) were quantitated with Elisa (Abazyme, Needham, MA) using serialdilutions of known amounts of HBsAg (9).

4. SiRNA Transfection

HepG2215 cells were seeded with the siRNAs (Dharmacon Inc., Lafayette,Colo.) at 50% density and incubated for 48 h. Subsequently, the cellswere washed and NIM811 or DEB025 was added (BL). Cellular andsupernatant samples were collected at 6 h, 24 h (medium was replaced)and at 48 hours. Total DNA was extracted from the samples and HBV DNAwas quantitated by real time PCR, as above. Total RNA was extracted withphenol, chloroform. The RNA was reverse transcribed with QuantitectReverse transcription kit (Qiagen) and cDNA quantitated with SYBR GreenQuantitect kit using Quantitect primers (Qiagen, Sussex, UK).

5. Immunoblot Analysis for HBsAg

After 48 hours, the cells (PLC/PRF/5 or HepG2215 cells) were trypsinizedand washed two times in phosphate-buffered saline and resuspended inlysis buffer. The presence of human IgG and HBsAg in PLC/PRF/5 cells wasanalyzed by western blot in cells cultured in the presence or absence ofmonoclonal anti-HBs IgG, as described.(7)

6. In Vitro Transcription for Hepatits Delta RNA

HDV RNAs 1.2 times the genome length are transcribed from plasmidspBSδ1.2G, pBSδ1.2AG, pBSδ1.2G(2×S), and pBSδ1.2AG(2×S) with T7MEGAscript kits (Ambion) after linearization with restriction enzymeNotI, as described previously (13). Capped mRNA for HDAg is transcribedfrom plasmid pX9-I/II after linearization with HindIII by using a T7m-Message m-Machine kit (Ambion). Unlabeled monomer genomic andantigenomic HDV RNAs are transcribed from pTMδSalA and pTMδSalB with T7MEGAscript after linearization by PstI digestion.

7. Northern Analysis of HDV RNA

Viral RNA from inocula or sera are purified using a QIAamp Viral RNAmini kit (QIAGEN, Valencia, Calif.) according to the manufacturer'sinstructions (14,15). Various amounts of RNA are then incubated at 55°C. for 50 min in the presence of 1.9 M glyoxal (Fisher Scientific, FairLawn, N.J.)-7.1 mM sodium phosphate (pH 6.8)-4.5 mM EDTA-35% DMSO.Samples are then loaded on a 1.5% agarose gel containing 10 mM sodiumphosphate, pH 6.8, and subjected to electrophoresis for 4 h at 150 mA.For supernatants, half of the RNA is used, while for cellular RNA, 5 μgis loaded. RNAs are capillary transferred overnight to Zeta-Probe(Bio-Rad, Richmond, Calif.) membranes. After transfer, the membrane iseither baked or UV cross-linked using a Stratalinker (Stratagene). Theblot is hybridized, as decided in detail (15). After hybridization, theblot is washed at 70° C. with 400 ml of 2× SSPE-0.1% SDS, then with 400ml of 1× SSPE-0.1% SDS, and then with 200 ml of 0.1× SSPE-0.1% SDS. Themembrane is dried at 70° C. and subjected to autoradiography andphosphorimager (Molecular Dynamics) analysis.

8. Real-Time PCR Quantification in Serum Samples (as DescribedPreviously (16))

HDV RNAs are extracted from 250 μl of serum or plasma by use of a QIAampMinElute virus vacuum (QIAGEN, Courtabceuf, France). cDNAs aresynthesized as previously described and are purified with Montage PCRcentrifugal filter devices (Millipore, Molsheim, France).

The forward primer is selected to target the ribozyme region of thegenome, and the reverse primer targeted region I of the antigenomeribozyme. The probe, which hybridizes to the same region as the reverseprimer, is designed to anneal to the antigenomic sequence to avoid basepairing with the reverse primer. The names and sequences of the primersand probe are as follows: Delta-F (forward primer),5′-GCATGGTCCCAGCCTCC-3′; Delta-R (reverse primer),5′-TCTTCGGGTCGGCATGG-3; and Delta-P (probe),5′-FAM-ATGCCCAGGTCGGAC-MGB-3′. Because of the existence of one mismatchwith the sequences of HDV-3 genomes, the following second direct primeris specifically designed for the amplification of HDV-3 isolates:T3-Delta-F, 5′-GCATGGCCCCAGCCTCC-3′.

Real-time PCRs are performed by using the TaqMan Universal PCR mastermix (Applied Biosystems, Courtabceuf, France). The reaction consists ofone initiating step of 2 min at 50° C., followed by 10 min at 95° C. andthen 45 cycles of amplification including 15 s at 95° C. and 1 min at60° C. The reactions, data acquisition, and analyses are performed withthe ABI PRISM 7000 sequence detection system (Applied Biosystems,Courtabceuf, France).

References:

1. Naoumov N V. Hepatitis Viruses (excluding hepatitis C virus). OxfordTextbook of Medicine 5^(th) Edition, Eds. D. Warrell, T. Cox, J. Firth,Oxford University Press, 2010, Vol 1: 609-615.

2. EASL Clinical Practice Guidelines: Management of chronic hepatitis B.J Hepatol 2009; 50:227-242.

3. Lok A, Mcmahon B. Chronic Hepatitis B: Update 2009. AASLD PracticeGuidelines. Hepatology 2009;50 (3):1-36.

4. Liaw Y -F et al. Asian-Pacific consensus statement on the managementof chronic hepatitis B: a 2008 update. Hepatol Int 2008.

5. Wedemeyer H, Manns M P. Epidemiology, pathogenesis and management ofhepatitis D: update and challenges ahead. Nat Rev Gastroenterol Hepatol.2010;7(1):31-40.

6. Wedemeyer H, et al. Peginterferon plus adefovir versus either drugalone for hepatitis delta. N Engl J Med. 2011;364(4):322-31.

7. R. Schilling, et al. Endocytosis of hepatitis B immune globulin intohepatocytes inhibits the secretion of hepatitis B virus surface antigenand virions. J Virol. 2003;77:8882-92.

8. S. Phillips, et al. CD8(+) T cell control of hepatitis B virusreplication: direct comparison between cytolytic and noncytolyticfunctions. J Immunol. 2010;184:287-95.

9. A. Neumann, et al. Novel mechanism of antibodies to hepatitis B virusin blocking viral particle release from cells. Hepatology2010;52:875-85.

10. Chao, M., S. -Y. Hsieh, and J. Taylor. 1990. Role of two forms ofthe hepatitis delta virus antigen: evidence for a mechanism ofself-limiting genome replication. J. Virol. 64:5066-5069.

11. Modahl, L. E., and M. M. C. Lai. 1998. Transcription of hepatitisdelta antigen mRNA continues throughout hepatitis delta virus (HDV)replication: a new model of HDV RNA transcription and regulation. J.Virol. 72:5449-5456.

12. Macnaughton T B, Lai M. Hepatitis Delta Virus RNA Transfection forthe Cell Culture Model Methods in Molecular Medicine, 2004, Volume 96,II, 351-357, DOI: 10.1385/1-59259-670-3:351

13. Macnaughton, T B et al. Rolling Circle Replication of HepatitisDelta Virus RNA Is Carried Out by Two Different Cellular RNA PolymerasesJ Virol. 2002; 76(8): 3920-3927.

14. Dingle, K., V. Bichko, H. Zuccola, J. Hogle, and J. Taylor. 1998.Initiation of hepatitis delta virus genome replication. J. Virol.72:4783-4788.

15. Bruno B. et al. A Prenylation Inhibitor Prevents Production ofInfectious Hepatitis Delta Virus Particles J Virol. 2002; 76(20):10465-10472.

16. Le Gal, F et al. Quantification of Hepatitis Delta Virus RNA inSerum by Consensus Real-Time PCR Indicates Different Patterns ofVirological Response to Interferon Therapy in Chronically InfectedPatients J Clin Microbiol. 2005; 43(5): 2363-2369.

1. Alisporivir for use in the treatment of Hepatitis B virus infectionand Hepatitis D virus infection in a patient.
 2. Alisporivir for useaccording to claim 1, wherein alisporivir is administered in combinationwith a direct antiviral agent or an interferon.
 3. A method for treatinga Hepatitis B virus infection in a patient comprising administering aneffective amount of alisporivir and optionally administering to thepatient an interferon or a direct antiviral agent.
 4. A method fortreating a Hepatitis Delta virus infected infection in a patientcomprising administering an effective amount of alisporivir andoptionally administering to the patient an interferon or a directantiviral agent.
 5. A method for prevention of progression of liverdisease in a patient comprising administering alisporivir.
 6. Apharmaceutical composition comprising alisporivir for use according toclaim 1.